Transforming growth factor-[3 (TGF-[3) is a potent inhibitor of human mammary epithelial proliferation. During mammary carcinogenesis most breast cancer cells become resistant to the growth inhibitory effects of TGF-13; TGF-13 then promotes breast cancer tumor formation. Fundamental gaps in knowledge exist in terms of the mechanisms (1) by which breast cancers become TGF-13 resistant and (2) by which TGF-[3 functions as both a tumor suppressor and a tumor promoter during mammary carcinogenesis. The type III TGF-[3 receptor (T13RIII) has an emerging yet poorly understood role in regulating TGF-[3 signaling and mammary carcinogenesis. Based on preliminary studies establishing the scaffolding protein 13-arrestin2 as a T13RlII- interacting protein, the following hypothesis is proposed: Interaction of T13RIII with 13-arrestin2 during mammm 7 carcinogenesis mediates (1) the internalization and downregulation of TGF-[3 receptors, resulting in resistance to TGF-[3-mediated inhibition of proliferation and (2) stimulation of T13RIII-dependent signaling to MAP kinase pathways promoting tumor formation. This hypothesis will be addressed by four Specific Aims. Specific Aim 1: The T[3RIII/[3-arrestin2 interaction will be characterized and the effects of Tf3RIII )hosphorylation and T[3RIII-interacting proteins (GIPC, Tt3RII) on the T13RIII/13-arrestin2 interaction and 13- m'estin2-mediated T13RIII internalization in human breast cancer cells will be established. Specific Aim 2: The effect of 13-arrestin2 on T13RIII expression, downregulation, TGF-I3 signaling and TGF-_-mediated inhibition of proliferation in human breast cancer cells will be established to define whether Tl3RIII/[3-arrestin2 mediates the tumor suppressor effects of TGF-[3 during mammary carcinogenesis. Specific Aim 3: The effect of 13- arrestin2 on T13RIII-dependent signaling to MAP kinase pathways and on the proliferation, motility, survival and anchorage-independent growth of human breast cancer cells will be established to define whether Tf3RIIl/13-arrestin2 mediates the tumor promoting effects of TGF-[3 during mammary carcinogenesis. Specific Aim 4: The effect of altering [3-arrestin2 or GIPC expression or the ability of T13RIII to interact with 13- arrestin2 or GIPC on the balance of T13RIII-mediated Smad-dependent and Smad-independent signaling and on the tumorigenicity/metastatic potential of breast cancer cells in vivo will be established to determine whether TI3RIII through [3-arrestin2 regulates TGF-I3 signaling during mammary carcinogenesis. These studies will define mechanisms for TGF-13-resistance, for Smad-independent signaling and for the dichotomous role of TGF-13 in mammary carcinogenesis and aid in targeting the TGF-13 signaling pathway for the chemoprevention and treatment of human breast cancers and other human cancers in which TGF-13 has a defined role.